The chromogenic assay used to monitor the potency of factor VIII does not consistently give the same result as a plasma substrate-based test. The standard measurement of factor VIII coagulant activity at the FDA employs the plasma substrate assay, while the European Pharmacopeia mandates a chromogenic test. Potency discrepancies between these two tests affect product fill and patient care. Furthermore, the replacement of plasma-derived products with recombinant equivalents has led to additional concerns about the comparability of these products and the applicability of current assay systems. Therefore, new testing procedures and/or molecular-based testing methods need to be developed in order to reliably assay both plasma-derived and recombinant-technology products. To understand the fundamental mechanisms and resolve the disparities between the tests and to develop assay methods for these products, we are evaluating molecular parameters that influence the assays and attempting to develop analytical methods based on surface plasmon resonance (SPR) biosensor technology measured by BIAcore. We have investigated the interaction kinetics and catalytic activities of rFVIII products when complexed with factor IXa on a supported hybrid bilayer membrane (HBM) to determine the role of phospholipids on the coagulation process. We have found that a HBM can be stably formed on the HPA sensor chip using unilamellar vesicles generated by reverse-phase evaporation of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine and 1-palmitoyl-2-oleoyl-sn-glycero-3-[phospho-L-serine]. Our data also indicated that various coagulation factors interacted strongly and reversibly with the HBM. The association and dissocation rate constants of different recombinant FVIII products tested were dissimilar. The binding of rFVIII products was inhibited in a concentration-dependent manner by an antibody to the light chain of the FVIII molecule. Furthermore, our data indicated that the molecular complex of rFVIIIa and FIXa, the tenase complex, was generated on the HBM and retained enzymatic activities.